Optical microresonators confine light to small volumes by resonant recirculation and have demonstrated potential use as microscopic light emitters, lasers, and sensors (K. J. Vahala, Nature 424, pp. 839-846, 2003). The recirculation imposes geometry-dependent boundary conditions on wavelength and propagation direction of the light kept inside the microresonator. Accordingly, only certain optical modes, the so-called “cavity modes”, can be efficiently excited. Since the energy levels of these allowed modes depend crucially on geometry and optical properties of the microresonators, the latter comprise very sensitive microscopic optical sensors that can be used for example to sense forces (e.g. by deformation of the cavity, cf., e.g., M. Gerlach et al., Opt. Express 15, 6, pp. 3597-3606, 2007) or changes in chemical concentration (e.g. by a corresponding change of the refractive index in close vicinity of the microresonator, cf., e.g., A. M. Armani, K. J. Vahala, Opt. Lett. Vol. 31, pp. 1896-1898, 2006). Similarly, microresonators can be used for biomolecular detection, e.g. by absorption of specifically binding molecules to or into a microresonator and detecting the resultant change of the refractive index around or inside of the cavity.
The confinement of light inside of a microresonator requires a highly reflective boundary between the microresonator and its surrounding. This can be achieved for example via total internal reflection (TIR), similarly to the guidance of light inside of an optical waveguide. As shown in FIG. 1A, TIR can occur if the refractive index of the microresonator, ncav, is larger than that of its surrounding, nenv, i.e. ncav>nenv. However, even in this case, TIR occurs only for angles α above a so-called “critical angle” αcrit=arcsin(nenv/ncav), where α is measured from the local surface normal inside of the cavity, where the reflection occurs (FIG. 1A). Such simple considerations remain valid as long as surface roughness is negligible as compared to the wavelength of the light stored inside the microresonator. Accordingly, one general lower size limit of microresonators is given by the precision to which smooth surfaces can be prepared.
Another obstacle for utilization of microresonators is directly related to the requirement of a highly reflective interface between microresonator and surrounding. Since the path of light is reversible in absorption-free media, the interface will be also highly reflective for those light beams that impinge onto the interface from the surrounding. Accordingly, just those optical modes inside the cavity, which fulfill the requirement of high reflectivity and thus provide high light storage potential, cannot be easily populated by light accessing the microresonator from the outside.
Vollmer and coworkers (F. Vollmer et al., Applied Physics Letters 80, pp. 4057-4059, 2002) used evanescent field coupling between the uncoated core of an optical fiber and a silica microsphere for population of the cavity modes inside of the microsphere. In this case, photons can transit from the high refractive index core of the fiber to the high refractive index interior of the microsphere via tunneling. However, it has been demonstrated by Z. Guo et al. (Journal of Physics D: Applied Physics 39, pp. 5133-5136, 2006) that the coupling efficiency as well as the frequencies of the generated cavity modes within the cavity are highly depending on the distance between the optical fiber and the cavity. As a consequence, both the microsphere and the optical fiber have to be fixed to a solid mount in order to keep the distance between them constant. Vollmer et al. were able to demonstrate cavity mode biosensing via adsorption of Bovine Serum Albumin (BSA) onto the outer surface of silica spheres with diameters of 300 μm. They showed that the sensitivity of their sensor scales with 1/R, where R is the particle radius.
Kuwata-Gonokami and coworkers (M. Kuwata-Gonokami et al., Jp. J. Appl. Phys. Vol. 31, pp. L99-L101, 1992) used dye-doped polystyrene (PS) microspheres for populating cavity modes. The microspheres were radiated with ultrashort laser pulses to excite the dye molecules. The pump laser pulse was incident onto the microsphere surface at a small incidence angle α, so that the light could penetrate into the optically denser microsphere with small loss only (typically 5-10%). The excited dye molecules inside of the microresonator re-radiate fluorescent light into arbitrary directions, i.e. also into those which fulfill the condition of total internal reflection. Accordingly, all cavity modes, which fell into the emission wavelength range of the dye molecules, became excited. At high pump intensities lasing was observed.
Woggon and coworkers (M. V. Artemyev and U. Woggon, Applied Physics Letters 76, pp. 1353-1355, 2000; B. Möller et al., Applied Physics Letters 80, pp. 3253-3255, 2002) used semiconductor quantum dots for doping of polymer latex beads. Similar to the work of Kuwata-Gonokami and coworkers, the cavity modes inside of the latex beads can be populated by excitation of the semiconductor quantum dots with light of suitable wavelength. The quantum dots then re-emit fluorescent light that excites those cavity modes within their emission range. In general, the emission bandwidth of quantum dots amounts to some tens of nanometers, i.e. it is smaller than that of most dye molecules. One major advantage of quantum dots is, however, their much higher stability with respect to photobleaching. Recently, Woggon and coworkers used this scheme also for the excitation of cavity modes in coupled microresonators (B. M. Möller et al., Optics Letters 30, pp. 2116-2118, 2005).
Halas and coworkers have suggested core-shell particles of much smaller size consisting of a non-metallic core and a metallic shell for optical biosensing (West et al., U.S. Pat. No. 6,699,724 B1). They studied in particular the size regime from few tens to several hundreds of nanometers, i.e. particles with an outer diameter <1 μm. The conductive shell of such particles can be optically excited at the so-called “plasma frequency”, which corresponds to a collective oscillation of the free electrons of the shell. While the plasma frequency of solid metal particles shows only marginal dependence on the particle size and is basically given by the physical properties of the bulk material, such as electron density and effective electron mass, Halas et al. were able to demonstrate that in the case of core-shell particles the position of the plasma frequency can be tuned over a wide range from the visible to the near infrared solely by changing the ratio between core and shell radii of the particles (N. Halas, Optics & Photonics News 13, 8, pp. 26-31, 2002; S. J. Oldenburg et al., Chemical Physics Letters 288, pp. 243-247, 1998). Halas et al. suggested to use such particles as biosensors by tuning the plasma frequency into a frequency range where it could support surface enhanced Raman emission of organic molecules adsorbed on the outer shell surface. The Raman emission then can serve as qualitative measure of protein adsorption. It must be noted, that Halas et al. used the core-shell character of the fabricated particles solely for tuning of the plasma frequency but not for generation or utilization of microresonator modes. In the course, they have not suggested to embed any kind of fluorescent material into the non-metallic particle cores for population of such modes.
A variety of cavity geometries has been studied so far. The most simple ones are microspheres, such as used by Vollmer et al. (F. Vollmer et al., Applied Physics Letters 80, pp. 4057-4059, 2002), rings or cylinders (D. K. Armani et al., Nature 421, pp. 925, 2003; H. J. Moon et al., Optics Communications 235, pp. 401, 2004). More complex cavities with lower degree of symmetry can also be used for the excitation of cavity modes, such as nanocrystals with hexagonal cross section (T. Nobis et al, Physical Review Letters 93, 10, 103903, 2004) or asymmetric cavities (Nöckel et al., Nature 385, pp. 45, 1997). Scherer and coworkers (O. Painter et al., Science 284, pp. 1819-1821, 1999) utilized photonic crystal structures and achieved the so far smallest microresonator volumes of 0.03 cubic micrometers with a single defect in a two-dimensional photonic crystal.
Several groups studied optical cavity mode spectra of assemblies of microresonators, such as dimers (T. Mukaiyama et al., Phys. Rev. Lett. Vol. 82, pp. 4623-4626, 1999), trimers and tetramers (B. M. Möller et al., Phys. Rev. B Vol. 70, pp. 115323/1-5, 2004), and linear chains (M. Bayer et al., Phys. Rev. Lett. Vol. 83, pp. 5374-5377, 1999; V. N. Astratov et al., Appl. Phys. Lett. Vol. 85, pp. 5508-5510, 2004; B. M. Möller et al., Opt. Lett. Vol. 30, pp. 2116-2118, 2005), solely in air and not for the purpose of optical sensing, but instead for cavity quantum electrodynamic studies and the development of coupled-resonator optical waveguides. In contrast to the present embodiment, most of these works utilized microresonators of same size and geometry (e.g. microstructured by means of lithographic patterning (Bayer et al.) or size-selected colloidal spheres (Mukaiyama et al., Möller et al.) with exactly matching cavity mode spectra to allow tight coupling and mode splitting, which is not observable otherwise (cf. e.g. Mukaiyama et al.). The utilization of microresonators of same geometry and size for the formation of assemblies of microresonators jeopardizes the idea of a characteristic spectral fingerprint for identification of a cluster of microresonators within an ensemble of clusters as implemented by the present embodiment. In fact, it is one of the key ideas of the present embodiment to take advantage of the size distribution of microresonators brought about by their fabrication and to utilize this variety for the generation of characteristic spectral fingerprint spectra. Further, Astratov and coworkers (V. N. Astratov et al., Appl. Phys. Lett. Vol. 85, pp. 5508-5510, 2004) studied optical coupling and transport phenomena in chains of spherical dielectric microresonators with a slight size dispersion of 1%. The chains consisted of a chain of non-fluorescent PS beads with one fluorescent bead at the top. The WGM emission of this bead was then traced down through the chain. As shown in FIG. 2 of that article, while the intensity drops from bead to bead with increasing distance from the light emitting bead for different modes to different extent, the observable WGM spectrum is always that of a single sphere, i.e. the non-fluorescing beads to not influence the WGM emission in terms of resonance positions or bandwidths. Effects of utilizing different chains on the appearance of the WGM spectrum of the light-emitting bead are not reported. Thus, conclusions about the existence of spectral fingerprint spectra in clusters of beads, in particular with larger size dispersion, cannot be drawn. More recently, Ashili et al. (S. P. Ashili et al., Opt. Express Vol. 14, pp. 9460-9466, 2006) studied optical coupling of cavity modes between two microspheres with large radius mismatch (8 and 5 μm diameters, respectively) as a function of their separation. While a slight blue shift of the WGM positions with decreasing gap size between the two microresonators was observed, this shift was attributed to a substrate effect and not to the sphere-sphere interaction. Therefore, no indication of the presence of spectral fingerprints is given. Summarizing, the studies found in the literature on optical cavity mode spectra of assemblies of microresonators do not give any evidence for the existence of spectral fingerprints as implemented in the present embodiment nor do they discuss the application of assemblies or clusters of microresonators for optical sensing applications.
For biosensing applications by means of optical cavity mode tracking in microresonators, so far mainly non-metallic microresonators have been applied. Ilchenko & Maleki (Proceedings SPIE, 4270, pp. 120-130, 2001) have described a set-up for using whispering gallery mode resonators with very high quality factors as optical sensors by monitoring the decrease of the quality factor due to molecular adsorption on the resonator surface. This principle requires very high quality factors in the range Q˜108−109, which are achievable only in resonators of several tens to several hundreds micrometers in diameter. Smaller resonators have typically much higher losses, and accordingly, lower quality factors. Therefore, the approach suggested here is not well suited for the development of an optical sensor with a total size of less than few tens of micrometers.
Maleki et al. (U.S. Pat. No. 6,490,039 B2) have described how to use a single microparticle with a spherical shape for biosensing. The detection is based on the cavity mode wavelength shift that occurs when a (bio-)molecule is adsorbed on the microsphere surface. The experimental setup requires a single microparticle with a high quality factor, which will host the cavity modes and act as a transducer. The cavity modes are generated by TIR within the microparticle using an incoming laser coupled to the microparticle by an optical fiber. The output signal is also collected by an optical fiber and then analyzed. The wavelength shift of the cavity modes provides information whether the (bio-) molecule is attached to the microparticle or not.
Poetter et al. (PCT/AU2005/000748, 2005) have applied a similar approach for biosensing by means of cavity modes. Following the approach of Woggon and coworkers, they have used fluorescent microparticles or particles that contain quantum dots. In that case, the cavity modes are excited by emission of the fluorophore inside of the sphere so that coupling between the microsphere and an optical fiber is not required. This approach further enables the use of different types of light sources (UV lamp, HeNe gas laser, Argon ion laser, HeCd gas laser, etc.) for the excitation of the fluorophore and thus the cavity modes.
Noto et al. (Applied Physics Letters 87, pp. 223901-1 223901-3, 2005; Biophysical Journal 92, pp. 4466-4472, 2007) have shown that biosensors based on cavity modes can be used not only in order to detect the presence of biomolecules attached on the surface of a microsphere but also in order to get some relevant information about the biomolecule itself. The authors have shown that the wavelength shift depends on the molecular weight of the biomolecule considered. The authors also pointed out that it is possible to determine the orientation of the biomolecule attached to the microsphere by comparing the cavity mode wavelength shift of different kinds of cavity mode excitations (transverse electric (TE) and transverse magnetic (TM) modes). The latter experiments have been performed with Bovine Serum Albumin (BSA) adsorbed onto the surface of a silica sphere (r=200 μm).
Vollmer et al. (Biophysical Journal 85, pp. 1974-1979, 2003), have described a biosensor for the detection of DNA based on the detection of cavity modes in single silica microspheres. The authors used two single microspheres (r=200 μm) that were functionalized with oligonucleotides in order to interact specifically with different nucleic acids. They demonstrated the multiplexed detection of specific DNA sequences by applying two microspheres coupled to a common optical fiber. In contrast to the present embodiment, the microspheres were operated independently of each other to assure an independent sensor signal. While the WGM resonance positions were detected through the same fiber, they were independently traced. In particular, no cross-coupling between the two microspheres, which were placed several micrometers apart from each other, was observed (p. 1976, 1st column, 3rd line from bottom). Further, the authors did not report about any specific differences in the spectra obtained from different sets of microspheres, i.e. they did not report about the existence of spectral fingerprints as defined in the present embodiment.
Teraoka et al. (Journal of Optical Society of America B 23, 7, pp. 1434-1441, 2006) have more recently described how to improve the sensitivity of biosensors based on cavity modes. The authors have coated a silica microsphere with a layer of higher refractive index material, in that case polystyrene. The authors have claimed a significant improvement in sensitivity towards biomolecular detection with this coated microsphere.
A number of groups have applied non-metallic optical microresonators as sensors in liquid environment, for example for remote refractive index sensing (P. Zijlstra et al., Appl. Phys. Lett. Vol. 90, pp. 161101/1-3, 2007; S. Pang et al. Appl. Phys. Lett. Vol. 92, pp. 221108/1-3, 2008; J. Lutti et al., Appl. Phys. Lett. 93, 15113/1-3, 2008). None of these groups however, have considered or investigated the optical properties and/or the application of clusters of microresonators.
Other groups have achieved lasing in microcavities, i.e. the fabrication and utilization of microlasers (e.g. M. Kuwata-Gonkami & K. Takeda, Opt. Mater. Vol 9, pp. 12-17, 1998; V. Sandoghar et al. Phys. Rev. A Vol. 54, pp. R1777-R1780, 1996; S. M. Spillane et al., Nature Vol. 415, pp. 621-623, 2002; Z. Zhang et al., Appl. Phys. Lett. 90, 111119/1-3, 2007). The achievement of lasing in clusters of microresonators, in particular in liquid environment and/or for biosensing application, however, has not been reported so far.
Summarizing, the work utilizing non-metallic microresonators for sensing applications has so far only been performed by utilization of isolated microresonators. In the case that multiplexing is discussed, i.e. the application of more than a single microresonator for parallel detection of a variety of analytes, the different microresonators applied are thought to be operated independently of each other.
The application of optical cavity modes of metal-coated dielectric particles to biosensing is described in WO2007129682. There, also clusters of metal-coated dielectric particles are described. However, there is no mention throughout the text that cavity mode spectra obtained from clusters of resonators may exhibit a characteristic fingerprint, which may be used for their recognition and/or an facilitated readout process.
Besides closed microresonators, also the utilization of open microresonators has been suggested for biosensing. These microresonators comprise microscopic vacancies in a thin metallic film. The light is confined only in the plane of the thin film, but free in perpendicular direction. Blair and coworkers (Y. Liu et al., Nanotechnology, 15, pp. 1368-1374, 2004; Y. Liu & S. Blair, Proceedings of SPIE, 5703, pp. 99-106, 2005) studied fluorescent enhancement of dye-labeled proteins adsorbed inside of nanocavities patterned in a thin gold film. They observed fluorescent enhancement by a factor of 2 and an increase in quantum yield by a factor of 6. While the authors have utilized assemblies of nanofabricated cavities basically to increase signal intensity, they have not spectrally analyzed the fluorescence emission obtained from their samples. Therefore, they were not able to observe neither optical cavity modes in general nor any characteristic spectral fingerprints as notably described in the present embodiment. In addition, the existence of such characteristic fingerprints is very unlikely in their case for two reasons. Firstly, the method relies on regular patterns, secondly, it applies electron beam lithography for their fabrication. Due to the excellent precision of this technique, deviations from the regularity of the pattern are expectedly small, thus jeopardizing the occurrence of spectral fingerprints, which are based on local deviations from the basic (regular) pattern.
There exist a variety of other methods for label-free biosensing based on plasma excitations of metal particles or thin metal films. In these cases, an incoming light wave is used to launch a freely propagating or localized surface plasmon (which corresponds to a collective oscillation of the free electrons of the metal). The plasmon in turn produces an evanescent electromagnetic wave in the close environment of the metal film or metal particle. When the dielectric properties in this environment are altered, e.g. due to biomolecular adsorption, the plasmon resonance position is changed. Accordingly, this shift can be used as read-out signal of a label-free optical biosensor. Examples of approaches utilizing localized plasmon effects are given in US 2003/0174384 A1, EP 0 965 835 A2, WO2006111414, Sensors and Actuators B Vol. 63, pp. 24-30, 2000, and Biosensors & Bioelectronics Vol. 22, pp. 3174-3181, 2007. WO2006111414 mentions explicitly the use of metal-coated clusters of fluorescent dielectric particles for biosensing. However, the use and/or excitation of optical cavity modes is neither discussed nor even mentioned.
An example for utilization of free-travelling plasmons is given by the Biacore system from General Electric Health Care, UK.
Recently, some groups discussed coupling between optical cavity modes and surface plasmons in single metal-coated particles (D. Amarie et al., Journal of Physical Chemistry B, Vol. 109, pp. 15515-15519, 2005) or in regular arrays of particles embedded in a metallic matrix (R. M. Cole et al., Physical Review Letters, Vol. 97, pp. 137401/1-4, 2006). The latter group did not report about characteristic spectral fingerprints, since they worked with periodic arrays of particles and could neglect the presence of imperfections. Further, their approach is not well suited for optical sensing, because surface plasmons are generated at the particle/metal interface, which is not easily accessible from the outside.